mmg_233_2013_genetics_genomicswikiaorg-20200214-history
The Ti Plasmid: Lateral Gene Transfer Mechanism and Utility
Crown gall is a common disease that affects many woody shrubs (especially those within the rose family) and also several genera of herbaceous plants. This disease, which results in the formation of an amorphous tumorous mass, is caused by Agrobacterium tumefaciens, a Gram-negative soil bacterium that can persist for many years in soils with good aeration (i.e. sandy loams) in a free-living state. Agrobacterium tumefaciens is capable of infecting over 140 genera, representing 93 families, of plants and is nearly ubiquitous in nature (5). What makes agrobacterium so unique is the mechanism by which it infects these plants: conjugative horizontal gene transfer. Agrobacterium and Crown Gall Disease Agrobacterium tumefaciens is an aerobic, Gram-negative bacterium that is often found at the plant-root inter-phase, a region that is sometimes termed the "rhizosphere". The microbe is also non-fastidious and of baciliform or rod-shaped morphology. The capsule of Agrobacterium is studded with multiple fimbriae and flagella, the later being arranged subpolary around the cylindrical circumference of the cell. When a A. tumefaciens bacterium is in close proximity to an injured susceptible plant, various virulent genes within the resident Ti (tumor inducing) plasmid are activated, resulting in the formation of a conjugative filament called the T pilus. The expression of these virulent genes also renders the bacterium's flagella inert. This is essential if the T-DNA within the Ti plasmid is to be transferred via the T pilus into the plant cell's nucleus as it would be impossible for the Agrobacterium to preform conjugative horizontal gene transfer if it were still in motion (2, 5). If a Agrobacterium strain is to be virulent, it must possess functional circumthecal flagella as these are necessary to move the bacterium into the vicinity of a susceptible plant, but above all else it must contain a Ti plasmid as this extra-chromosomal element directs the formation of the T pilus necessary for horizontal gene transfer and contains the oncogenic T-DNA that will be inserted into the plant to induce gall formation. Several chromosomal elements, including chvA and chvB genes, are also essential to Agrobacterium virulence (2). Many of these chromosomal elements are regulate Ti plasmid gene expression, though they are also known to promote signal molecule recognition and play a role in Agrobacterium adhesion. The chvA and chvB genes in particular are known to be responsible for the specificity by which Agrobacteria bind to plant cells and are constitutively expressed (2, 5). Crown gall disease only results if the plant of interest has been wounded as the T pilus is unable to penetrate the cellulose of an intact cell wall. Gall formation can disrupt the normal flow of water and essential nutrients through the phloem of affected plants, resulting in reduced crop yields and stunted growth. Though fresh gall tumors are quite hardy, galls aged more than a year are typically friable and riddled with cavities harboring parasitic insects. These older galls generally do not contain many living Agrobacteria as they are composed primarily of deceased, diseased tissue (5). Ti Plasmid A residual Ti or "Tumor inducing" plasmid can be found in all virulent Agrobacterium strains. This large extrachromosomal DNA element contains 196 genes and over 200 kilobases. The virulence and T-DNA regions of the Ti plasmid are of particular interest when one is discussing the conjugative, pathogenic properties of this plasmid (5, 6). Though only the T-DNA will ever enter the plant cell, genes within the virulence region of the Ti plasmid encode the polypeptide sequences that will modulate and ultimately lead to the transduction of T-DNA into the vulnerable plant cell. The Ti plasmid also facilitates its own conjugal transfer to other bacterial cells lacking a Ti plasmid. The initiation of conjugation in A. tumefaciens is tightly regulated by opines, small chemical compounds characteristically found within the neoplastic growths that form as a result of Crown Gall disease. The enzymes required for the synthesis of these low molecular weight compounds are encoded within the T-DNA of the Ti plasmid; it is after the T-DNA has integrated into the plant's genome that opines are actively synthesized by the infected plant (3, 4). Virulence Region The virulence region of the Ti plasmid contains 7 operons of interest. These operons include virA, virB, virC, virD1, virD2, virE, and virG. The virA operon encodes various dimethoxyl phenolic compound receptors, a feature that allows the bacterium to detect and respond to the presence of injured plant tissues as wounded plants will exude these chemicals into their immediate environments. The virB operon encodes pilus proteins that play a pivotal role in T pilus formation. Expression of the virG operon results in the synthesis of a transcription factor that will be termed the virG transcription factor for convenience. The dimethoxyl phenolic compound receptors encoded by the virA operon are histidine kinases that will phosphorylate the virG transcription factor after encountering a specific phenolic compound. This now activated virG transcription factor will subsequently induce the transcription of the remaining operons within the virulence region. Vir A activation also forestalls flagellum motility, rendering the bacterium immobile while the T pilus forms. One of the protein products of the virD2 operon accompanies the T-DNA across the T-pilus while other virD2 (and virD1) gene products serve as endonucleases and catalyze the cleavage of T-DNA at the repeat border sequences (5, 6). The expression of different operons in the virulence region of the Ti plasmid is variable, depending on the operon in question. Only the virA operon is consistently expressed, thought the virG region goes through periods of constitutive expression. All other elements within the virulence operon are inducible, requiring a phosphorylated and thus activated vir G transcription factor to be expressed. The 2 largest operons within the virulence region (operons virB and virD) are likely transcribed as polycistronic messenger RNA. Though all operons play a key role in Agrobacterium pathogenesis, only mutations within the 5 operons described (virA, virB, virD1, virD2 and virG) will completely eliminate the ability of Agrobacterium tumefaciens to induce neoplastic changes in a plant cell (2). T-DNA T-DNA or "transfer DNA" denotes all Ti plasmic DNA that passes through the T-pilus from Agrobacterium tumefaciens to the plant cell to be infected. T-DNA is transferred as a single stranded DNA molecule chaperoned by a virD2 protein into the nucleus of the plant cell where it integrates into one of the plant's chromosomes (5). The later part of this statement has been affirmed by numerous in-situ hybridization experiments. Within T-DNA, there are 5 regions of interest including the left and right borders and 3 anabolic genes encoding the biosynthesis of auxin, cytokinin and select opines. The right and left borders contain repetitive DNA sequences that allow for the selective cleavage and subsequent transportation of T-DNA (6). Once the T-DNA integrates into the plant's genome via non-homologous recombination, the plant's transcriptional machinery will synthesize the phytohormones auxin and cytokinin, eliminating the cell's dependence on exogenous hormones and resulting in the prolific, neoplastic growth characteristic of Crown Gall disease. The integration of T-DNA also results in the biosynthesis of various opine metabolites which serve a carbon and nitrogen sources for Agrobacterium tumefaciens. The production of certain opines (i.e. octopine and agrocinopine) also promotes the conjugative transfer of Ti plasmids between the Agrobacterium tumefaciens microbes present (3,4, 5). Utility in Modern Science The Ti plasmid of Agrobacterium tumefaciens has been used in plant genetic engineering for several decades now, a fact that accentuates is immense utility in this role. The ability of the Ti plasmid to induce neoplastic growth must be expunged before it can be of any use, however. As these neoplastic properties derive mainly from the Ti plasmid's anabolic auxin and cytokinin genes, these genes are often replaced with the foreign DNA the researcher wishes to integrate into the plant's genome. A genetic marker such as an antibiotic resistance or green fluorescent protein gene, is also introduced into the engineered Ti plasmid so that the researchers can later identify and isolate all cells that have been successfully transformed (1). General procedure Once the DNA fragment of interest has been cloned and inserted into a viable Ti plasmid, the engineered plasmid is inserted into an Agrobacterium tumefaciens bacterium. This gene of interest is often accompanied by a gene conferring antimicrobial resistance so that cells that have been successfully transformed can be isolated with ease later on. The genitally modified Agrobacterium are then allowed to infect cultured cells, leaf discs or root slices (1). Though not explicitly mentioned in the text I consulted, I believe that the integrity of the plant cell walls must be compromised in some way (possibly electroporation) before the bacteria are introduced if the plants are to become transgenic. The infected cells are then cultured in a selective medium containing auxin, cytokinin and the antibiotic the engineered plasmid confers resistance to, a chemical cocktail that will induce the growth of all transformed cells while inhibiting the growth of all cells that haven't been genetically modified. A whole plant can be obtained from transformed cells by simply adjusting the ratio of phytohormones in the culture medium to encourage shoot and root formation (1). References 1. The Ti Plasmid and Plant Genetic Engineering 2. The genetic and transcriptional organization of the Ti plasmid vir region 3. Chemistry and biochemistry of opines 4. Regulation of Agrobacterium Ti plasmid conjugal transfer 5. Crown Gall Disease 6. Ti Plasmid